This proposal describes research designed to extend understanding of two recent developments concerning the control of hepatic glucose metabolism by epinephrine and glucagon. Recent evidence suggests that, in rat liver, epinephrine and glucagon regulate glycogen metabolism and gluconeogenesis via separate mechanisms, glucagon via the agency of cyclic AMP and epinephrine via an alpha receptor mechanism that is independent of cyclic AMP. Other developments point to a growing understanding that these hormones need to act in concert at several enzyme sites within the cell to effectively stimulate gluconeogenesis. The objectives of the proposed research are twofold. The first objective is to determine how important control of each of the known regulated sites in the gluconeogenic pathway is to effective stimulation of gluconeogenesis by epinephrine and/or glucagon. This objective will be approached by stimulating isolated liver cells with catecholamines and glucagon and comparing the time course and magnitude of the hormone effects on the enzymes presently thought to be regulated during gluconeogenesis. Initial attention will be focused on cyclic AMP and the activities of pyruvate carboxylation in the mitochondria and pyruvate kinase and fructose diphosphatase in the cytoplasm. The second objective will be to determine if the cytoplasmic control points in the gluconeogenic pathway are regulated by cycles of enzyme phosphorylation and dephosphorylation. It is proposed to focus in detail on one of the control sites, pyruvate kinase, and to determine if epinephrine, acting through an alpha mechanism and glucagon acting through cyclic AMP, regulate this enzyme's activity via such a cycle. This objective will be approached by incubating isolated liver cells with radioactive phosphate, stimulating the cells with the hormones and following the incorporation of radioactive phosphate into pyruvate kinase. The labeled proteins and the enzymes will be separated by polyacrylamide slab gel electrophoresis.